Arabidopsis was engineered to produce 21.2 % punicic acid in the seed oil. Possible molecular factors limiting further accumulation of the conjugated fatty acid were investigated. Punicic acid (18:3Δ(9cis,11trans,13cis) ) is a conjugated linolenic acid isomer and is a main component of Punica granatum (pomegranate) seed oil. Medical studies have shown that punicic acid is a nutraceutical with anti-cancer and anti-obesity properties. It has been previously demonstrated that the conjugated double bonds in punicic acid are produced via the catalytic action of fatty acid conjugase (FADX), which is a homolog of the oleate desaturase. This enzyme catalyzes the conversion of the Δ(12)-double bond of linoleic acid (18:2Δ(9cis,12cis) ) into conjugated Δ(11trans) and Δ(13cis) -double bonds. Previous attempts to produce punicic acid in transgenic Arabidopsis thaliana seeds overexpressing P. granatum FADX resulted in a limited accumulation of punicic acid of up to 4.4 %, accompanied by increased accumulation of oleic acid (18:1∆(9cis) ), suggesting that production of punicic acid in some way inhibits the activity of oleate desaturase (Iwabuchi et al. 2003). In the current study, we applied a new strategy to enhance the production of punicic acid in a high linoleic acid A. thaliana fad3/fae1 mutant background using the combined expression of P. granatum FADX and FAD2. This approach led to the accumulation of punicic acid at the level of 21 % of total fatty acids and restored the natural proportion of oleic acid observed in the A. thaliana fad3/fae1 mutant. In addition, we provide new insights into the high oleate phenotype and describe factors limiting the production of punicic acid in genetically engineered plants.